A marked resistance to the stimulatory action of insulin on glucose metabolism has previously been shown in guinea pig compared to rat adipocytes. We have examined the mechanism of insulin resistance in guinea pig adipoctyes by measuring a variety of parameters including insulin binding, the stimulatory effect of insulin on lipogenesis and the stimulatory effect of insulin on glucose transport. Based on these studies we present evidence that demonstrates that the poor responsiveness of guinea pig adipocytes to insulin is due to a reduction in the number of glucose carrier proteins. We have used a photosensitive crosslinking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), to covalently attach 3H-cytochalasin B to a rat adipocyte low density microsomal protein of molecular weight 45-50,000. The labeling of this protein was sensitive to preincubation of membranes with D- but not with L-glucose. The amount of 3H-cytochalasin B incorporated into the membrane proteins varied depending on the metabolic state of the cells (control or insulin treated) prior to homogenization. Isoelectric focusing (IEF) of the SDS-PAGE purified photoaffinity crosslinked protein yielded 3 peaks of radioactivity. The major peak at pH 5.4 was isolated and found to crossreact with antibodies raised against purified erythrocyte glucose transporter. Based on these studies we strongly suggest that the 45-50K protein that we have photoaffinity labeled with cytochalasin B is the adipocyte glucose transport carrier.